US20060160208A1 - Multi-well plate with alignment grooves for encoded microparticles - Google Patents

Abstract

A method and apparatus are provided for aligning optical elements or microbeads, wherein each microbead has an elongated body with a code embedded therein along a longitudinal axis thereof to be read by a code reading device. The microbeads are aligned with a positioning device so the longitudinal axis of the microbeads is positioned in a fixed orientation relative to the code reading device. The microbeads are typically cylindrically shaped glass beads between 25 and 250 microns (μm) in diameter and between 100 and 500 μm long, and have a holographic code embedded in the central region of the bead, which is used to identify it from the rest of the beads in a batch of beads with many different chemical probes. A cross reference is used to determine which probe is attached to which bead, thus allowing the researcher to correlate the chemical content on each bead with the measured fluorescence signal. Because the code consists of a diffraction grating typically disposed along an axis, there is a particular alignment required between the incident readout laser beam and the readout detector in two of the three rotational axes. The third axis, rotation about the center axis of the cylinder, is azimuthally symmetric and therefore does not require alignment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• This application is a continuation-in-part of, and relates and claims benefit to the following U.S. provisional patent application Ser. No. 60/546,435 (CV-0053PR), entitled “Multi-Well Plate With Alignment Grooves for Encoded Particles”, filed Feb. 19, 2004, which is hereby incorporated by reference in their entirety.
• This application also relates to U.S. provisional patent application Ser. Nos. 60/546,445 (CV-0035PR), entitled “Optical Identification Element Having Non-waveguide Photosensitive Substrate with Bragg Grating Therein”; and60/547,013 (CV-0065PR), entitled “Optical Identification Element Using Separate or Partially Overlapping Diffraction Gratings”, all filed Feb. 19, 2004 and hereby incorporated by reference in their entirety, as well as their corresponding United States patent applications (CV-0035US and CV-0065US), as well as any corresponding PCT patent applications, all filed on this day contemporaneously with the instant application and all also hereby incorporated by reference in their entirety.
• The following cases contain subject matter related to that disclosed herein and are incorporated herein by reference in their entirety: U.S. Provisional Patent Application Ser. No. 60/441,678, filed Jan. 22, 2003, entitled “Hybrid Random Bead/Chip Microarray”, (Attorney Docket No. CC-0574); U.S. patent application Ser. No. 10/645,689, filed Aug. 20, 2003, entitled “Diffraction Grating-Based Optical Identification Element”, (Attorney Docket No. CC-0648); U.S. patent application Ser. No. 10/645,686 filed Aug. 20, 2003, entitled “End Illuminated Bragg Grating based Optical Identification Element”, (Attorney Docket No. CC-0649); U.S. patent application Ser. No. 10/661,234, filed Sep. 12, 2003, entitled “Diffraction Grating-Based Optical Identification Element”, (Attorney Docket No. CC-0648A); U.S. patent application Ser. No. 10/661,031, filed Sep. 12, 2003, entitled “End Illuminated Bragg Grating based Optical Identification Element”, (Attorney Docket No. CC-0649A); U.S. patent application Ser. No. 10/661,082, filed Sep. 12, 2003, entitled “Method and Apparatus for Labeling Using Diffraction Grating-based Encoded Optical Identification Elements”, (Attorney Docket No. CC-0650); U.S. patent application Ser. No. 10/661,115, filed Sep. 12, 2003, entitled “Assay Stick”, (Attorney Docket No. CC-0651); U.S. patent application Ser. No. 10/661,836, filed Sep. 12, 2003, entitled “Method and Apparatus for Aligning Microbeads in order to Interrogate the Same”, (Attorney Docket No. CC-0652); U.S. patent application Ser. No. 10/661,254 filed Sep. 12, 2003, entitled “Chemical Synthesis Using Diffraction Grating-based Encoded Optical Elements”, (Attorney Docket No. CC-0653); U.S. patent application Ser. No. 10/661,116 filed Sep. 12, 2003, entitled “Method of Manufacturing of a Diffraction grating-based identification Element”, (Attorney Docket No. CC-0654); U.S. Provisional Patent Application Ser. No. 60/519,932, filed Nov. 14, 2003, entitled, “Diffraction Grating-Based Encoded Microparticles for Multiplexed Experiments”, (Attorney Docket No. CC-0678); and U.S. patent application Ser. No. 10/763,995 filed Jan. 22, 2004, entitled, “Hybrid Random bead/chip based microarray”, (Attorney Docket No. CV-0054).
BACKGROUND OF THE INVENTION
• 1. Technical Field
• The present invention generally relates to a method and apparatus for processing information contained on microbeads, each microbead having an elongated body with a code embedded therein along a longitudinal axis thereof to be read by a code reading device; and more particularly to a method and apparatus for aligning the microbeads so the longitudinal axis thereof is in a fixed orientation relative to the code reading or other device.
• 2. Description of Related Art
• Many industries have a need for uniquely identifiable objects or for the ability to uniquely identify objects, for sorting, tracking, and/or identification/tagging. Existing technologies, such as bar codes, electronic microchips/transponders, radio-frequency identification (RFID), and fluorescence and other optical techniques, are often inadequate. For example, existing technologies may be too large for certain applications, may not provide enough different codes, or cannot withstand harsh temperature, chemical, nuclear and/or electromagnetic environments.
• It has long been understood that RT-PCR is capable of providing an industry leading assay sample throughput so long as the required number of measurements per sample is limited to less than about 6 samples. On the other end of the spectrum, microarrays have a low sample throughput but are capable of providing industry leading numbers of measurements per sample (upwards to 100,000 samples). There is a strong desire in the industry for a technology that can offers both high sample throughput and a large number of measurements per sample. Several technologies have been proposed to meet this challenge. One example is printing microarrays in the bottom of wells of microtitre plates (GeneXP, Matrix). This technology is attractive because it preserves the microtitre format necessary for high sample throughput, but is limited in the number of spots that can be printed. Gene XP, for example, can print a maximum of 1024 spots. As they use 3 probe replicates (spots) per measurement, GeneXP is limited to a total of 340 measurements per sample. Other technologies that attempt to solve this problem include eTag (Aclara), fluorescent microbeads (Luminex, Quantum Dot), spatially ordered microbeads (Illumina), molecular multiplexed profiling (HGT), and others.
• Therefore, it would be desirable to obtain a coding element or platform that provides the capability of providing many codes (e.g., greater than 1 million codes), that can be made very small and/or that can withstand harsh environments.
• Moreover, it would be desirable to provide a method and apparatus to position and align such coding elements so as to better sense the chemical content and correlate it in relation to the code to determine information about the process.
SUMMARY OF THE INVENTION
• In its broadest sense, the present invention provides a new and unique method and apparatus for aligning new and unique coding elements or microbeads, wherein each microbead has an elongated body with a code embedded therein along a longitudinal axis thereof to be read by a code reading or other detection device. The method features the step of aligning the microbeads with a positioning device so the longitudinal axis of the microbeads is positioned in a fixed orientation relative to the code reading or other detection device.
• The new and unique microbeads are not spherical, but instead have an elongated shape and may be cylindrical, cubic, rectangular, or any other elongated shape. The microbeads are typically composed of silica glass with some germanium and/or boron doped region or regions that are photosensitive to ultraviolet light. Coded microbeads are individually identifiable via a single or series of spatially overlapping pitches written into them. The microbeads may be used in many different processes. After such processing, the microbeads have a resulting chemical content on the surface of each bead that is sensed and correlated in relation to the code contained with the microbead to determine information about the process.
• When used in an assay process, the microbeads are typically cylindrically (i.e. tubular) shaped glass beads and between 25 and 250 μm in diameter and between 100 and 500 μm long. They have a holographic code embedded in the central region of the bead, which is used to identify it from the rest of the beads in a batch of beads with many different DNA or other chemical probes. A cross reference is used to determine which probe is attached to which bead, thus allowing the researcher to correlate the chemical content on each bead with the measured fluorescence signal. Because the code consists of a diffraction grating typically disposed along an axis of the microbead, there is a particular alignment required between the incident readout laser beam and the readout detector in two of the three rotational axes. In aeronautical terms, the two of the three rotational axes include the pitch of the microbead in the front-to-back direction and the yaw of the microbead in a side-to-side direction. The third axis, rotation about the center axis of the cylinder, is azimuthally symmetric and therefore does not require alignment. The third axis is analogous to the roll axis.
• The invention provides a method for aligning the microbeads in the two rotational axes to a fixed orientation relative to an incident laser beam and a readout camera, otherwise known as the code camera. The invention further provides a method for rapidly aligning a large number of microbeads, between 1,000 and 1,000,000 microbeads or more, economically, and with the necessary tolerances. The method is flexible as it relates to the size of the microbeads and can be integrated into a fully automated system, which prepares the microbeads for rapid readout by an automated code-reading machine.
• In one embodiment of the present invention, the positioning device includes a plate with a series of parallel grooves, which could have one of several different shapes, including square, rectangular, v-shaped, semi-circular, etc., as well as a flat bottom groove with tapered walls. The grooves are formed into an optically transparent medium such as borosilicate glass, fused silica, or plastic. The depth of the grooves will depend on the diameter of the microbead but generally they will be between 10 and 125 μm, but may be larger as discussed hereinafter depending on the application. The spacing of the grooves is optimal when it is between 1 and 2 times the diameter of the microbead, providing for both maximum packing density as well as maximum probability that a microbead will fall into a groove. The width of grooves is optimal when the gap between the microbead and the walls of the grooves is sufficiently small to prevent the microbeads from rotating within the grooves by more than a few degrees. The bottom of the groove must also be maintained flat enough to prevent the microbeads from rotating, by more than a few tenths of a degree, relative to the incident laser beam. Another critical aspect of the grooved plate is the optical quality of the grooves. To prevent excess scatter of the readout laser beam, which could lead to low contrast between the code signal and the background scatter, it is important that the grooves exhibit high optical quality. The beads can be read in the groove plate from the bottom of (i.e. below), from the top of (i.e. above), or from the side of the plate, depending on the application and type of microbead used.
• Some advantages of the groove plate approach include:
• Rapid simultaneous alignment of microbeads. Alignment rates ˜1000's per second.
• Once the microbeads are aligned, they can be read as many times as necessary to get a good reading or improve statistics.
• Microbeads naturally fall into groove (presumably by capillary forces) at very high packing densities.
• Microbeads can be mixed after reading then re-read to enhance the statistics of readout process.
• In an alternative embodiment of the present invention, the positioning device may includes a tube having a bore for receiving, aligning and reading the microbeads.
• Moreover, the present invention also provides an apparatus for aligning an optical identification element. The optical identification element having an optical substrate having at least a portion thereof with at least one diffraction grating disposed therein, the grating having at least one refractive index pitch superimposed at a common location, the grating being embedded within a substantially single material, the grating providing an output optical signal when illuminated by an incident light signal, the optical output signal being indicative of a code, and being the result of passive, non-resonant scattering from said at least one diffraction grating when illuminated by said incident light signal, and the optical identification element being an elongated object with a longitudinal axis. The apparatus also having an alignment device which aligns the optical identification element such that said output optical signal is indicative of the code.
• The present invention also provides an optical element capable of having many optically readable codes. The element has a substrate containing an optically readable composite diffraction grating having one or more collocated index spacing or pitches Λ. The invention allows for a high number of uniquely identifiable codes (e.g., millions, billions, or more). The codes may be digital binary codes and thus are digitally readable or may be other numerical bases if desired.
• Also, the elements may be very small “microbeads” (or microelements or microparticles or encoded particles) for small applications (about 1-1000 microns), or larger “macroelements” for larger applications (e.g., 1-1000 mm or much larger). The elements may also be referred to as encoded particles or encoded threads. Also, the element may be embedded within or part of a larger substrate or object.
• The code in the element is interrogated using free-space optics and can be made alignment insensitive.
• The gratings (or codes) are embedded inside (including on or near the surface) of the substrate and may be permanent non-removable codes that can operate in harsh environments (chemical, temperature, nuclear, electromagnetic, etc.).
• The code is not affected by spot imperfections, scratches, cracks or breaks in the substrate. In addition, the codes are spatially invariant. Thus, splitting or slicing an element axially produces more elements with the same code. Accordingly, when a bead is axially split-up, the code is not lost, but instead replicated in each piece.
• The foregoing and other objects, features and advantages of the present invention will become more apparent in light of the following detailed description of exemplary embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWING
• The drawing is not drawn to scale and includes the following Figures:
• FIG. 1 shows steps of a microbead platform assay process.
• FIG. 2 is a side view of an optical identification element in accordance with the present invention.
• FIG. 3 is a top level optical schematic for reading a code in an optical identification element in accordance with the present invention.
• FIG. 4 is a perspective view of a grooved plate for use with an optical identification element, in accordance with the present invention.
• FIG. 5 is a diagram of the flat grooves and an example of the dimensionality thereof in accordance with the present invention.
• FIG. 6 is a perspective view of a plate with holes for use with an optical identification element, in accordance with the present invention.
• FIG. 7 is a perspective view of a grooved plate for use with an optical identification element, in accordance with the present invention.
• FIG. 8 is a diagram of a microbead mapper reading, in accordance with the present invention.
• FIG. 8ais a diagram of a system for both detecting a material on and reading a code in a microbead, in accordance with the present invention.
• FIG. 9 is a diagram of a plate having microbeads thereon in relation to an open plate format for detection and reading of the microbead in accordance with the invention.
• FIG. 10 is a diagram of a starting point for handling microbeads for readout in a cuvette process in accordance with the invention.
• FIG. 11 is a diagram of a second step in the readout process in accordance with the invention.
• FIG. 12 is a diagram of the readout step in accordance with the invention.
• FIG. 13 is a diagram of final steps in the cuvette process in accordance with the invention.
• FIG. 14 is a diagram of an example of the cuvette showing its mount on a kinematic plate in accordance with the invention.
• FIG. 15 is a diagram of an alternative embodiment of a cuvette showing a port for fluid filling/emptying using a pipette in accordance with the invention.
• FIG. 16 is a diagram of an alternative embodiment of a cuvette showing an alternative port for fluid filling/emptying using a pipette in accordance with the invention.
• FIG. 17 is a diagram of a two zone cuvette showing a free region and a trapped region in accordance with the invention.
• FIG. 18(a) is a diagram of steps for a conventional flow cytometer reader in a single pass cytometer process in accordance with the invention.
• FIG. 18(b) is a diagram of steps for a disk cytometer reader in a multipass cytometer process in accordance with the invention.
• FIGS.19(a), (b) and (c) show embodiments of a disk cytometer in accordance with the invention.
• FIG. 20(a) show an embodiment of a disk cytometer having radial channels for spin drying in accordance with the invention.
• FIG. 20(b) show an alternative embodiment of a disk cytometer having a mechanical iris for providing a variable aperture for bead access to grooves in accordance with the invention.
• FIG. 21 show an embodiment of a SU8 groove plate having 450 in accordance with the invention.
• FIG. 21 show an embodiment of a SU8 cylindrical grooved plate having 450×65 microns beads in accordance with the invention.
• FIG. 22 show an embodiment of an alignment tube in accordance with the invention.
• FIG. 23 show an alternative embodiment of an alignment tube having a receiving flange in accordance with the invention.
• FIG. 24 is an optical schematic for reading a code in an optical identification element, in accordance with the present invention.
• FIG. 25(a) is an image of a code on a CCD camera from an optical identification element, in accordance with the present invention.
• FIG. 25(b) is a graph showing an digital representation of bits in a code in an optical identification element, in accordance with the present invention.
• FIG. 26 illustrations (a)-(c) show images of digital codes on a CCD camera, in accordance with the present invention.
• FIG. 27 illustrations (a)-(d) show graphs of different refractive index pitches and a summation graph, in accordance with the present invention.
• FIG. 28 is an alternative optical schematic for reading a code in an optical identification element, in accordance with the present invention.
• FIG. 29, illustrations (a)-(b), are graphs of reflection and transmission wavelength spectrum for an optical identification element, in accordance with the present invention.
• FIGS. 30-31 are side views of a thin grating for an optical identification element, in accordance with the present invention.
• FIG. 32 is a perspective view showing azimuthal multiplexing of a thin grating for an optical identification element, in accordance with the present invention.
• FIG. 33 is side view of a blazed grating for an optical identification element, in accordance with the present invention.
• FIG. 34 is a graph of a plurality of states for each bit in a code for an optical identification element, in accordance with the present invention.
• FIG. 35 is a side view of an optical identification element where light is incident on an end face, in accordance with the present invention.
• FIGS. 36-37 are side views of an optical identification element where light is incident on an end face, in accordance with the present invention.
• FIG. 38, illustrations (a)-(c), are side views of an optical identification element having a blazed grating, in accordance with the present invention.
• FIG. 39 is a side view of an optical identification element having a coating, in accordance with the present invention.
• FIG. 40 is a side view of whole and partitioned optical identification element, in accordance with the present invention.
• FIG. 41 is a side view of an optical identification element having a grating across an entire dimension, in accordance with the present invention.
• FIG. 42, illustrations (a)-(c), are perspective views of alternative embodiments for an optical identification element, in accordance with the present invention.
• FIG. 43, illustrations (a)-(b), are perspective views of an optical identification element having multiple grating locations, in accordance with the present invention.
• FIG. 44 is a perspective view of an alternative embodiment for an optical identification element, in accordance with the present invention.
• FIG. 45 is a view an optical identification element having a plurality of gratings located rotationally around the optical identification element, in accordance with the present invention.
• FIG. 46, illustrations (a)-(e), show various geometries of an optical identification element that may have holes therein, in accordance with the present invention.
• FIG. 47, illustrations (a)-(c), show various geometries of an optical identification element that may have teeth thereon, in accordance with the present invention.
• FIG. 48, illustrations (a)-(c), show various geometries of an optical identification element, in accordance with the present invention.
• FIG. 49 is a side view an optical identification element having a reflective coating thereon, in accordance with the present invention.
• FIG. 50, illustrations (a)-(b), are side views of an optical identification element polarized along an electric or magnetic field, in accordance with the present invention.
• FIGS. 51 and 52 are diagrams of beads read from flat retro-reflector trays, in accordance with the present invention.
• FIGS. 53 and 54 are diagrams of beads read through V-grooves, in accordance with the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
• FIG. 1 shows, by way of example, steps of a microbead assay platform process which uses the microbead technology of the present invention. The steps of the assay process shown inFIG. 1 include a first step in which the microbeads are used or hybridized in a solution; a second step in which the microbeads are aligned or self-assembled in a desired manner; a third step in which the code and florescence in and/or on the microbeads are read-out in solution; and a fourth step in which the information related to the code and florescence is processed in relation to data management and bioinformatics. The present invention primarily relates to step 2 wherein the microbeads are uniquely aligned so the longitudinal axis of the microbeads is positioned in a fixed orientation relative to the code and florescence reading device, as well as relating to a lesser extent to step 3. It is important to note that the scope of the present invention is not intended to be limited to any particular type or kind of assay process or other process in which the microbead technology is used. The scope of the invention is intended to include embodiments in which the microbead technology of the present invention is used in many different processes.
FIG.2: TheMicrobead Element8
• FIG. 2 shows a diffraction grating-based optical identification element8 (or encoded element or coded element) that comprises a knownoptical substrate10, having anoptical diffraction grating12 disposed (or written, impressed, embedded, imprinted, etched, grown, deposited or otherwise formed) in the volume of or on a surface of thesubstrate10 along the length or longitudinal axis L of theelement8, which is otherwise known hereinafter as the microbead. The grating12 is a periodic or aperiodic variation in the effective refractive index and/or effective optical absorption of at least a portion of thesubstrate10. Theoptical identification element8 described herein is the same as that described in copending patent application Ser. No. 10/661,032 (CiDRA Docket No. CC-0648A), filed Sep. 12, 2003, which is incorporated herein by reference in its entirety. It is important to note that the grating shown and described herein is provided by way of example. The scope of the invention is not intended to be limited to the type or kind of grating12 in the substrate, the type or kind of variations forming the same, or the manner or technique for disposing the grating12 into thesubstrate10. Moreover, the scope of the invention is intended to include gratings and techniques for disposing the same both now known in the art, as well as those developed in the future.
• In particular, thesubstrate10 has aninner region20 where the grating12 is located. Theinner region20 may be photosensitive to allow the writing or impressing of the grating12. Thesubstrate10 has anouter region18, which does not have the grating12 therein.
• The grating12 is a combination of one or more individual spatial periodic sinusoidal variations (or components) in the refractive index that are collocated at substantially the same location on thesubstrate10 along the length of thegrating region20, each having a spatial period (or pitch) Λ. The resultant combination of these individual pitches is the grating12, comprising spatial periods (Λ1-Λn) each representing a bit in the code. Thus, the grating12 represents a unique optically readable code, made up of bits, where a bit corresponds to a unique pitch Λ within thegrating12. Accordingly, for a digital binary (0-1) code, the code is determined by which spatial periods (Λ1-Λn) exist (or do not exist) in a givencomposite grating12. The code or bits may also be determined by additional parameters (or additional degrees of multiplexing), and other numerical bases for the code may be used, as discussed herein and/or in the aforementioned patent application. However, it is important to note that the scope of the invention is not intended to be limited to the type or kind of code represented by the grating12, or the manner or technique for reading or interpreting the same. Moreover, the scope of the invention is intended to include the grating represent codes, and/or or the manner or technique for reading or interpreting the same, both now known in the art, as well as those developed in the future.
• The grating12 may also be referred to herein as a composite or collocated grating. Also, the grating12 may be referred to as a “hologram”, as the grating12 transforms, translates, or filters an input optical signal to a predetermined desired optical output pattern or signal.
• Thesubstrate10 has an outer diameter D1 and comprises silica glass (SiO₂) having the appropriate chemical composition to allow the grating12 to be disposed therein or thereon. Other materials for theoptical substrate10 may be used if desired. For example, thesubstrate10 may be made of any glass, e.g., silica, phosphate glass, borosilicate glass, or other glasses, or made of glass and plastic, or solely plastic. For high temperature or harsh chemical applications, theoptical substrate10 made of a glass material is desirable. If a flexible substrate is needed, plastic, rubber or polymer-based substrate may be used. Theoptical substrate10 may be any material capable of having the grating12 disposed in thegrating region20 and that allows light to pass through it to allow the code to be optically read.
• Theoptical substrate10 with the grating12 has a length L and an outer diameter D1, and theinner region20 diameter D. The length L can range from very small “microbeads” (or microelements, micro-particles, or encoded particles), about 1-1000 microns or smaller, to larger “macro beads” or “macroelements” for larger applications (about 1.0-1000 mm or greater). In addition, the outer dimension D1 can range from small (less than 1000 microns) to large (1.0-1000 mm and greater). Other dimensions and lengths for thesubstrate10 and the grating12 may be used within the spirit of the invention described herein.
• The grating12 may have a length Lg of about the length L of thesubstrate10. Alternatively, the length Lg of the grating12 may be shorter than the total length L of thesubstrate10.
• Theouter region18 is made of pure silica (SiO₂) and has a refractive index n2 of about 1.458 (at a wavelength of about 1553 nm), and the innergrating region20 of thesubstrate10 has dopants, such as germanium and/or boron, to provide a refractive index n1 of about 1.453, which is less than that ofouter region18 by about 0.005. Other indices of refraction n1,n2 for thegrating region20 and theouter region18, respectively, may be used, if desired, provided the grating12 can be impressed in the desiredgrating region20. For example, thegrating region20 may have an index of refraction that is larger than that of theouter region18 orgrating region20 may have the same index of refraction as theouter region18 if desired.
FIG.3: The Code Reader orDetector29
• FIG. 3 shows a configuration for reading or detecting the code in themicrobead8 using a code reader orother detector device29, which is used instep 3 of the process shown inFIG. 1. In operation, anincident light24 of a wavelength λ, e.g., 532 nm from a known frequency doubled Nd:YAG laser or 632 nm from a known Helium-Neon laser, is incident on the grating12 in thesubstrate10. Any other input wavelength λ can be used if desired provided λ is within the optical transmission range of the substrate (discussed more herein and/or in the aforementioned patent application). A portion of the input light24 passes straight through the grating12, as indicated by anarrow25. The remainder of theinput light24 is reflected by the grating12, as indicated by anarrow27 and provided to adetector29. Theoutput light27 may be a plurality of beams, each having the same wavelength λ as the input wavelength λ and each having a different output angle indicative of the pitches (θ1-Λn) existing in thegrating12. Alternatively, theinput light24 may be a plurality of wavelengths and theoutput light27 may have a plurality of wavelengths indicative of the pitches (Λ1-Λn) existing in thegrating12. Alternatively, the output light may be a combination of wavelengths and output angles. The above techniques are discussed in more detail herein and/or in the aforementioned patent application. It is important to note that the scope of the invention is not intended to be limited to any particular input wavelength used, or the number of wavelengths used, or the number of beams used, or the angle of the beams used, in the technique for reading the code in theoptical identification element8 within the spirit of the invention.
• The code reader ordetector29 has the necessary optics, electronics, software and/or firmware to perform the functions described herein. In particular, the detector reads theoptical signal27 diffracted or reflected from the grating12 and determines the code based on the pitches present or the optical pattern, as discussed more herein or in the aforementioned patent application. An output signal indicative of the code is provided on aline31.
FIG.4: The Grooved Tray or Plate
• FIG. 4 shows one embodiment of apositioning device200 for aligning themicrobeads8 so the longitudinal axis of the microbeads is in a fixed orientation relative to the code reading or other detection device. Thepositioning device200 is shown in the form of a tray orplate200 having V-grooves205 for aligning themicrobeads8 and is used instep 2 of the process shown inFIG. 1.
• As shown, themicrobead elements8 are placed in thetray200 with V-grooves205 to allow theelements8 to be aligned in a predetermined direction for illumination and reading/detection as discussed herein. Alternatively, thegrooves205 may haveholes210 that provide suction to keep theelements8 in position.
Forming the Grooves in the Groove Plate
• The grooves in thegroove plate200 may be made in many different ways, including being formed by SU8 photoresistant material, mechanically machining; deep reactive ion etching; or injection molding. One advantage of the injection molding approach is that the plate can be manufactured in volume at relatively low cost, and disposed of after the information about the beads is gathered in the assay process. The groove plate may be made of glass, including fused silica, low fluorescence glass, borosilicate glass. Silicon is used because it is reflective so a reflective coating is typically not needed. Alternative, a mirror coating can be applied to the plate material to achieve the desired reflectivity. The scope of the invention is not intended to be limited the manner or technique for forming the grooves in the groove plate, and is intended to include manners and techniques both now known or later developed in the future.
FIG.5: Flat Grooves
• The scope of the invention is not intended to be limited to any particular groove shape. For example,FIG. 5 shows a diagram aplate300 having flat grooves302 instead of V-grooves as shown inFIG. 3. Some characteristics of the groove according to the present invention are as follows:
• The groove width (w) should be at least as wide as the diameter of the bead (D) but not larger than D+15 μm.
• The thickness of the depth of the groove (T) should be at least 0.5 times the diameter of the bead so that it sufficiently traps a bead once it falls into the groove even when it is subjected to mechanical agitation. The depth should not exceed 1.5 times the diameter of the bead so as to prevent more than one bead from falling into the same groove location.
• Groove plates have been made using a thick photoresist called SU8 and is available from Microchem. The resist is both chemically inert and mechanically robust once fully cured. The groove walls are formed by the resist material, which is deposited onto a glass or substrate. Advantages of this process include the ability to tailor the depth of groove by controlling the thickness of the resist material, and virtually every other geometric attribute through the design of the photo mask. Because it is photolithographic process, essentially any shape profile can be made. For example grooves can be made in simple rows, concentric circles, or spirals. Other features such as discrete wells, spots and cross hatches can be made as fiducial marks for tracking and positional registration purposes.
• The scope of the invention is also intended to include the grooves having a flat bottom as shown inFIG. 5 with outwardly tapered walls.
FIG.6: TheHoley Plate674
• FIG. 6 shows an alternative embodiment, wherein alignment may be achieved by using aplate674 havingholes676 slightly larger than theelements8 if the light24 (FIGS. 2 and 4) is incident along thegrating axis207. The incident light indicated as670 is reflected off the grating and exits through the end as a light672 and the remaining light passes through the grating and theplate674 as aline678. Alternatively, if a blazed grating is used,incident light670 may be reflected out the side of the plate (or any other desired angle), as indicated by aline680. Alternatively, input light may be incident from the side of theplate674 and reflected out the top of the plate474 as indicated by aline684. The light672 may be a plurality of separate light beams or a single light beam that illuminates theentire tray674 if desired.
FIG.7: V-Groove Plate200 with End Illumination
• FIG. 7 shows an alternative embodiment, wherein the V-groove plate discussed hereinbefore withFIG. 4 may be used for the end illumination/readout condition. In this case, the grating12 may have a blaze angle such that light incident along the axial grating axis will be reflected upward, downward, or at a predetermined angle for code detection. Similarly, the input light may be incident on the grating in a downward, upward, or at a predetermined angle and the grating12 may reflect light along the axial grating axis for code detection.
FIG.8: Microbead Mapper Readings
• FIG. 8 showsmicrobeads8 arranged on aplate200 havinggrooves205. As shown, themicrobeads8 have three different codes (e.g. “41101”, “20502”, “41125”) using 16-bit, binary symbology, which may be read or detected using the reader or detector configuration described in relation toFIG. 3. The codes in the beads are used to provide a cross reference to determine which probe is attached to which bead, thus allowing the researcher to correlate the chemical content on each bead with the measured fluorescence signal inStep 3 of the process shown inFIG. 1.
• FIG. 8ashows acode reader820 and adetector808 that form part of a device generally indicated as824 for obtaining information from themicrobead8 inFIG. 8. The codes in themicrobeads8 are detected when illuminated by incident light24 fromcode excit801 which produces a diffracted or outputlight signal27 to acode reader820, which includes the optics and electronics necessary to read the codes in eachbead8, as described herein and/or in the aforementioned copending patent application. Thecode reader820 provides a signal on aline822 indicative of the code in each of thebead8. Theincident light24 may be directed transversely from the side of the grooved plate200 (or from an end or any other angle) with a narrow band (single wavelength) and/or multiple wavelength source, in which case the code is represented by a spatial distribution of light and/or a wavelength spectrum, respectively, as described hereinafter and in the aforementioned copending patent application. Other illumination, readout techniques, types of gratings, geometries, materials, etc. may be used for themicrobeads8, as discussed hereinafter and in the aforementioned patent application.
• For assays that use fluorescent molecule markers to label or tag chemicals, anoptical excitation signal800 fromexcit source803 is incident on themicrobeads8 on thegrooved plate200 and a fluorescentoptical output signal802 emanates from thebeads8 that have the fluorescent molecule attached. The fluorescentoptical output signal802 passes through alens804, which provides focused light806 to a knownoptical fluorescence detector808. Instead of or in addition to thelens804, other imaging optics may be used to provide the desired characteristics of the optical image/signal onto thefluorescence detector808. Thedetector808 provides an output signal on aline810 indicative of the amount of fluorescence on a givenbead8, which can then be interpreted to determine what type of chemical is attached to thebead8.
• Consistent with that discussed herein, thegrooved plate200 may be made of glass or plastic or any material that is transparent to the codereading incident beam24 and code reading output light beams27 as well as thefluorescent excitation beam800 and the output fluorescentoptical signal802, and is properly suited for the desired application or experiment, e.g., temperature range, harsh chemicals, or other application specific requirements.
• Thecode signal822 from thebead code reader820 and thefluorescent signal810 from thefluorescence detector808 are provided to a knowncomputer812. Thecomputer812 reads the code associated with each bead and determines the chemical probe that was attached thereto from a predetermined table that correlates a predetermined relationship between the bead code and the attached probed. In addition, thecomputer812 reads the fluorescence associated with each bead and determines the sample or analyte that is attached to the bead from a predetermined table that correlates a predetermined relationship between the fluorescence tag and the analyte attached thereto. Thecomputer812 then determines information about the analyte and/or the probe as well as about the bonding of the analyte to the probe, and provides such information on a display, printout, storage medium or other interface to an operator, scientist or database for review and/or analysis, consistent with shown instep 4 ofFIG. 1. Thesources801,803, thecode reader820, thefluorescence optics804 anddetector808 and thecomputer812 may all be part of one device known as an assay stick reader, which in that case would be generally indicated as824.
• Alternatively, instead of having thecode excitation source801 and thefluorescence excitation source803, thedevice824 may have only one source beam which provides both the reflectedoptical signal27 for determining the code and thefluorescence signal802 for reading the tagged analyte attached to thebeads8. In that case, the input optical signal has a common wavelength that performs both functions simultaneously, or sequentially, if desired.
• Themicrobeads8 may be coated with the desired probe compound, chemical, or molecule prior to being placed in thegrooved plate200. Alternatively, thebeads8 may be coated with the probe after being placed in thegrooved plate200. As discussed hereinbefore, the probe material may be an Oligo, cDNA, polymer, or any other desired probe compound, chemical, cell, or molecule for performing an assay.
• The scope of the invention is not intended to be limited to using or detecting fluorescent molecule markers during the assay process. For example, embodiments of the invention are envisioned using and detection other types of molecular markers in other types of processes.
Modes of Microbead Alignment
• According to the present invention, there are at least two possible modes or approaches of use for the groove plate, as follows:
FIG.9: Open Format Approach
• FIG. 9 shows the first, or open plate format, meaning there is no top to cover themicrobeads8 and the V-grooves205. In this mode, themicrobeads8 are dispensed onto theplate200 using, for example, a pipette tip or syringe tip, although the scope of the invention is not intended to be limited to the manner of depositing the microbeads on the plate. Themicrobeads8 may be then agitated by a sonic transducer (not shown), or manipulated with a mechanical wiper (not shown) or some form of spray nozzle (not shown) to encourage all themicrobeads8 to line up in thegrooves205. It has been observed that substantially all the microbeads naturally line up in thegrooves205 without the need for encouragement. However, there are always some microbeads, such asmicrobead8a,8b, as shown, that do not fall naturally into the grooves, and these must either be removed from theplate200 or forced to fall into agroove205. The open format approach has the advantages that it consists of just theplate200 having grooves2005 and no other complicated features such as walls and a top, and possibly other chambers or channels to allow fluid flow and bubble removal. It also has the advantage that it can easily be made with a standard microscope slide, which is designed to fit all conventional microarray readers. However, the open format approach would most likely require the microbeads to be dried out prior to reading to prevent non-uniform and unpredictable optical aberrations caused by the uneven evaporation of the buffer solution.
FIGS.10-17: The Closed Format Approach
• FIGS. 10-17 show the second mode which is called a closed format, that consists of not only of a groove plate but also a top and at least three walls to hold the solution and the microbeads in a cuvette-like device generally indicated as500 as shown, for example, inFIG. 10.
• In summary, the closed format approach provides a method for effectively distributing and aligning microbeads during the readout process, as described below:
• The basic process for handling microbeads with a cuvette for readout consists of the following steps:
• (1)FIG. 10 shows a starting point for handling microbeads for a readout. Themicrobeads8 start in abead test tube502. Typical test-tube volumes are typically 1.5 ml, although the scope of the invention is not intended to be limited to any particular volume. Themicrobeads8 will generally be in a liquid (usually water with a small amount of other buffer chemicals to adjust pH and possibly a small amount [˜0.01%] of detergent.) As shown, thebead test tube502 contains themicrobeads8 in a solution generally indicated as507, which forms part ofstep 1 of the process shown inFIG. 1.
• (2)FIG. 11 shows thebead tube502 is coupled to aflange504 of thecuvette500, and thecuvette500 is inverted so the beads flow onto thegroove plate503. Thecuvette500 consists of two round flanges that accept test-tubes502,506, atransparent window501, and an opposinggroove plate503.FIG. 14 shows a drawing of a prototype cuvette. The groove plate outer dimensions can be any size, but typical microscope slide dimensions are convenient (e.g. 1″×3″). The grooves are mechanically or laser cut lengthwise, and have dimensions that are chosen for the exact size of cylindrical microbead. For instance, for a 125 μm diameter bead, grooves of approximately 150 μm wide by 150 μM deep are used. Onetube502 carries themicrobeads8 and a small amount of the carrier fluid orsolution507. Thesecond tube506 may be larger and hold more of the fluid. The purpose of thesecond tube506 is to guarantee a certain fluid level in the next step.
• (3) After the cuvette is inverted and the microbeads flow out onto the groove plate side of the cuvette, the microbeads naturally align in the grooves via a small amount of rocking or agitation, which forms part ofstep 2 of the process shown inFIG. 1.
• (4)FIG. 12 shows the readout step, in which, after thebeads8 are all (or nearly all) aligned in thegroove plate503, the entire plate is moved (or alternatively the readout laser beam may be scanned, or some combination thereof) in order to read the codes of each bead, which forms part ofstep 3 of the process shown inFIG. 1. In effect, once themicrobeads8 are in the grooves, theentire cuvette500 is moved back and forth across areadout beam508. Thereadout beam508 is transmitted through thecuvette500 and contains the code bits encoded on the scattering angles generally indicated as509.
• (5)FIG. 13 shows a final step, in which after the readout process thecuvette500 is inverted to its original position and thebeads8 flow back into theoriginal tube502, which forms part ofstep 3 of the process shown inFIG. 1.
• FIG. 14 shows an example of a cuvette generally indicated as700 that is mounted on akinematic base plate710. As shown, thecuvette700 has atube702 for holding the solution with the beads and atop window704 that is a 1 mm thick glass plate having dimensions of about 1″ by 3″. The cuvette also has a bottom plate that is a transparent groove plate. The location pins712 andlever arm714 hold thecuvette700 in place on thekinematic plate710.
• One of the key advantages of using the cuvette device is that the potential to nearly index match the glass microbeads with a buffer solution thereby reducing the divergence of the laser beam caused by the lensing effect of the microbeads, and minimizing scatter from the groove plate itself.
• Another advantage involves the potential to prevent microbeads from ever stacking up on top of each other, by limiting the space between the bottom and the top plate to be less than twice the diameter of the microbeads.
• Another advantage is that the cover keeps the fluid from evaporating.
FIGS.15-16
• FIGS. 15-16 show alternative embodiments of thecuvette500 shown inFIGS. 10-14. As shown, themicrobeads8 are injected from ahyperdermic needle2001 into acuvette2000,2000′ by placing them near the edge of anopening2002,2002′ and allowing the surface tension, or an induced fluid flow, to pull the microbeads into thecuvette2000,2000′, where, because of the limited height between the floor and the ceiling of thecuvette2000,2000′, they are confined to move around in a plane, albeit with all the rotational degrees of freedom unconstrained. Once in thecuvette2000,2000′, themicrobeads8 are quickly and sufficiently constrained by the grooves as themicrobeads8 fall into them. As in the case of the open format, there is still the finite probability that some number ofmicrobeads8 will not fall into the grooves and must be coaxed in by some form of agitation (ultrasonic, shaking, rocking, etc.). InFIG. 16, thecuvette2000′ has aport2003′ for topping off the fluid level.
FIG.17: Two Region Approach
• FIG. 17 shows an alternative embodiment of the closed approach, which involves sectioning the closed region into two regions, oneregion2010 where the microbeads are free to move about in a plane, either in a groove or not, and asecond region2012 where the microbeads are trapped in one ormore grooves2014 and can only move along the axes indicated by arrow G of agroove2014. Trapping themicrobeads8 in agroove2014 is accomplished by further reducing the height of the interior chamber to the extent that themicrobeads8 can no longer hop out of agroove2014. In this embodiment, thefree region2010 is used to pre-align themicrobeads8 into a groove, facilitating the introduction ofmicrobeads8 into the trappedsection2012. By tilting this type of cuvette up, gravity can be used to pull themicrobeads8 along agroove2014 from thefree region2010 to the trappedregion2012. Once in the trappedregion2012, themicrobeads8 move to anend2016 of thegroove2014 where they stop.Subsequent microbeads8 will begin to stack up until the groove is completely full ofmicrobeads8, which are stacked head to tail. This has the advantage of packing a large number of microbeads into a small area and prevents themicrobeads8 from ever jumping out of the grooves. This approach could also be used to align themicrobeads8 prior to injection into some form of flow cytometer, or a dispensing apparatus.
FIGS.18-23: The Cytometer
• FIGS. 18-23 show methods and apparatuses related to using a cytometer.
• FIG. 18(a) shows steps for a method related to a conventional (single pass) flow cytometer reader, andFIG. 18(b) shows a method related to a disk cytometer reader (multipass). InFIG. 18(a), the method generally indicated as900 has astep901 for providing beads and a solution similar to step 1 inFIG. 1; and a step902 for reading information from the beads similar tosteps 2 and 3 inFIG. 1. InFIG. 18(b), the method generally indicated as1000 has a step1001 for providing beads and solution similar to step 1 inFIG. 1; and astep1002 for spinning and reading information from the beads similar tosteps 2 and 3 inFIG. 1. In the methods shown in FIGS.18(a) and (b), a rotating disk1003 (see FIGS.19(a), (b) and (c) and20) is used for aligning the microbeads consistent withstep 2 of the process shown inFIG. 1.
• FIG. 19(a) shows an embodiment of a cytometer bead reader having a rotating disk generally indicated as1250, having adisk platform1252 with circumferential, concentric,grooves1254 for aligningmicrobeads8. As shown, therotating disk1250 has various sectors for processing the microbeads, including abead loading zone1256, abead removal zone1258 and areadout zone1260.
• FIG. 19(b) shows an alternative embodiment of a rotating disk generally indicated as1200, having adisk platform1202 withplanar groove plates1204a, b, c, d, e, fthat are shown with grooves oriented in any one or more different ways. One or more of theplanar groove plates1204a, b, c, d, e, fmay have an optional channel (SeeFIG. 20(a)) for fluid run-off, as shown, and a barrier for preventing the microbeads from flying off the plate. As shown, thewindow1262 for reading the beads is in contact with the fluid containing the beads.
• FIG. 19(c) shows an alternative embodiment of a rotating disk generally indicated as1280, having adisk platform1282 withradial grooves1284a,1284b. Thedisk platform1282 has abead loading zone1286 in the center of the disk. One advantage of this embodiment is that the opening of thebead loading zone1286 will also serve to allow the release of air bubbles that will naturally collect in the center of the disk due the reduced density of the fluid, which results from the centrifugal force pushing the fluid radially outwardly. Therotating disk1280 has tight bead packing due to the centrifugal forces due to the spinning action of the disk. Therotating disk1280 has awedge shape spacer1288 that keeps the channel at a constant gap width and awall1290.
• FIG. 20(a) shows an alternative embodiment of a rotating disk generally indicated as1300 having narrow radial channels1302 for spin drying so liquid is forced out of the circumferential grooves through the radial channels. Theplate1300 may have amechanical catcher1320 coupled thereto for moving radially outwardly indirection1320aif desired, for recirculating loose beads.
• FIG. 20(b) show an alternative embodiment of adisk cytometer1400 having amechanical iris1402 for providing a variable aperture for bead access to grooves in accordance with the invention.
• FIG. 21 shows arotating groove plate1404 having 450 by 65microns beads8 arranged in the rotating SU8circumferential channels1406, each channel having correspondingchannel walls1408,1410.
Continuous Mode—Process Steps
• Consistent with that shown and described above, the following are the processing steps for a continuous mode of operation:
• 1. Dispense batch of microbeads onto plate.
• 2. Spin slowly while agitating the plate theta x and y to get microbeads into grooves. The agitation can be performed using rocking, ultrasound, airflow, etc.
• 3. Once a sufficient number of microbeads are in grooves, spin up plate or disk to remove excess microbeads (microbeads that did not go into a groove).
• 4. Spin plate or disk to read code and fluorescence off microbeads.
• 5. To remove microbeads, purge with high velocity aqueous solution (enough to knock microbeads out of groove) and vacuum up, or spin microbeads off plate while they are not in a groove.
• 6. Inspect disk (probably with code camera) to verify that all microbeads have been removed.
• 7. Inject next batch of microbeads.
FIGS.22-23: TheAlignment Tube502
• InFIG. 22, instead of a flat grooved plate200 (FIG. 3), themicrobeads8 may be aligned in atube5020 that has a diameter that is only slightly larger than thesubstrate10, e.g., about 1-50 microns, and that is substantially transparent to theincident light24. In that case, theincident light24 may pass through thetube5020 as indicated by the light5000 or be reflected back due to a reflective coating on thetube5020 or the substrate as shown byreturn light504. Other techniques can be used for alignment if desired.
• FIG. 23 shows thetube5020 has anopening flange5120 for receiving themicrobeads8.FIG. 23 also shows anexcitation laser550, adiode laser552 and aCCD camera554 for gathering information from thebead8 consistent with that discussed above.
• FIGS.24-44: Reading the Microbead Code
• FIGS. 24-44 show, by way of example, a method and apparatus for reading the code in themicrobead8. The scope of the invention is not intended to be limited in any way to manner in which the code is read, or the method of doing the same.
• Referring toFIG. 24, the reflectedlight27 comprises a plurality of beams26-36 that pass through alens37, which provides focused light beams46-56, respectively, which are imaged onto aCCD camera60. Thelens37 and thecamera60, and any other necessary electronics or optics for performing the functions described herein, make up the reader ordetector29. Instead of or in addition to thelens37, other imaging optics may be used to provide the desired characteristics of the optical image/signal onto the camera60 (e.g., spots, lines, circles, ovals, etc.), depending on the shape of thesubstrate10 and input optical signals. Also, instead of a CCD camera, other devices may be used to read/capture the output light.
• Referring toFIG. 25(a), the image on theCCD camera60 is a series of illuminated stripes generally indicated byreference label120 indicating ones and zeros of a digital pattern or code of the grating12 in themicrobead8. The ones are indicated byreference labels122,124, . . . ,132. InFIG. 25(b), lines generally indicated byreference label68 on a graph generally indicated by70 are indicative of a digitized version of the image shown inFIG. 25(a) as indicated in spatial periods (Λ1-Λn), which corresponds to theones122,124, . . . ,132 shown inFIG. 25(a).
• Consistent with that shown inFIG. 24, each of the individual spatial periods (Λ1-Λn) in the grating12 is slightly different, thus producing an array of N unique diffraction conditions (or diffraction angles) discussed more hereinafter. When theelement8 is illuminated from the side bylight beam24 in the region of the grating12 at an appropriate input angle, e.g., of about 30 degrees as shown, with a single input wavelength λ (monochromatic) source, the diffracted (or reflected) beams26-36 are generated. Other input angles θi may be used if desired, depending on various design parameters as discussed herein and/or in the aforementioned patent application, and provided that a known diffraction equation (Eq. 1 below) is satisfied:
  sin(θ_i)+sin(θ_o)=mλ/nΛ  Eq. 1
  where Eq. 1 is diffraction (or reflection or scatter) relationship between input wavelength λ, input incident angle θi, output incident angle θo, and the spatial period Λ of the grating12. Further, m is the “order” of the reflection being observed, and n is the refractive index of thesubstrate10. The value of m=1 or first order reflection is acceptable for illustrative purposes. Eq. 1 applies to light incident on outer surfaces of thesubstrate10 which are parallel to the longitudinal axis of the grating (or the k_Bvector). Because the angles θi, θo are defined outside thesubstrate10 and because the effective refractive index of thesubstrate10 is substantially a common value, the value of n in Eq. 1 cancels out of this equation, as person skilled in the art would appreciate.
• Thus, for a given input wavelength λ, grating spacing Λ, and incident angle of the input light Λi, the angle Λo of the reflected output light may be determined. Solving Eq. 1 for θo and plugging in m=1, gives:
  θo=sin⁻¹(λ/Λ−sin(θi))  Eq. 2
  For example, for an input wavelength λ=532 nm, a grating spacing Λ=0.532 microns (or 532 nm), and an input angle of incidence θi=30 degrees, the output angle of reflection will be θo=30 degrees. Alternatively, for an input wavelength λ=632 nm, a grating spacing Λ=0.532 microns (or 532 nm), and an input angle θi of 30 degrees, the output angle of reflection θo will be at 43.47 degrees, or for an input angle θi=37 degrees, the output angle of reflection will be θo=37 degrees. Any input angle that satisfies the design requirements discussed herein and/or in the aforementioned patent application may be used, and the scope of the invention is not intended to be limited to any such input angle.
• In addition, to have sufficient optical output power and signal to noise ratio, theoutput light27 should fall within an acceptable portion of the Bragg envelope (or normalized reflection efficiency envelope)curve200, as indicated bypoints204,206 inFIG. 24, also defined as a Bragg envelope angle θB, as also discussed herein and/or in the aforementioned patent application. InFIG. 24, thecurve200 may be defined as:$\begin{array}{cc}I\left(\mathrm{ki},\mathrm{ko}\right)\approx {\left[\mathrm{KD}\right]}^2\sin {\mathrm{<figure-callout\; label="c"\; filenames="US20060160208A1-20060720-D00001.png,US20060160208A1-20060720-D00004.png"\; state="\{\{state\}\}">c</figure-callout>}}^2\left[\frac{\left(\mathrm{ki}-\mathrm{ko}\right)D}{2}\right]& \mathrm{Eq}.3\end{array}$
  where K=2πδn/λ, where δn is the local refractive index modulation amplitude of the grating and where λ is the input wavelength, sin c(x)=sin(x)/x, and where the vectors k_i=2π cos(θ_i)/λ and where k_o=27π cos(θ_o)/λ are the projections of the incident light and the output (or reflected) light, respectively, onto the line203 normal to the axial direction of the grating12 (or the grating vector k_B), D is the thickness or depth of the grating12 as measured along the line203 (normal to the axial direction of the grating12). Other substrate shapes than a cylinder may be used and will exhibit a similar peaked characteristic of the Bragg envelope. The inventors have found that a value for δn of about 10⁻⁴in the grating region of the substrate is acceptable; however, other values may be used if desired.
• Rewriting Eq. 3 gives the reflection efficiency profile of the Bragg envelope as follows:$\begin{array}{cc}I\left(\mathrm{ki},\mathrm{ko}\right)\approx {{\left[\frac{2\pi ·\delta n·D}{\lambda }\right]}^2\left[\frac{\mathrm{Sin}\left(x\right)}{x}\right]}^2& \mathrm{Eq}.4\end{array}$
  where: x=(ki−ko)D/2=(πD/λ)*(cos θi−cos θo)
• Thus, when the input angle θi is equal to the output (or reflected) angle θ_o(i.e., θi=θ_o), the reflection efficiency I (Eqs. 3 & 4) is maximized, which is at the center or peak of the Bragg envelope. When θi=θo, the input light angle is referred to as the Bragg angle as is known. The efficiency decreases for other input and output angles (i.e., θi≠θ_o), as defined by Eqs. 3 & 4. Thus, for maximum reflection efficiency and thus output light power, for a given grating pitch Λ and input wavelength, the angle θi of theinput light24 should be set so that the angle θo of the reflected output light equals the input angle θi.
• Also, as the thickness or diameter D (FIG. 2) of the grating12 decreases, the width of the sin(x)/x function (and thus the width of the Bragg envelope) increases and, the coefficient to or amplitude of the sin c²(or (sin(x)/x)²function (and thus the efficiency level across the Bragg envelope) also increases, and vice versa. Further, as the wavelength λ increases, the half-width of the Bragg envelope as well as the efficiency level across the Bragg envelope both decrease. Thus, there is a trade-off between the brightness of an individual bit and the number of bits available under the Bragg envelope. Ideally, δn should be made as large as possible to maximize the brightness, which allows D to be made smaller.
• From Eq. 3 and 4, the half-angle of the Bragg envelope θ_Bis defined as follows:$\begin{array}{cc}{\theta }_B=\frac{\eta \lambda }{\pi D\sin \left({\theta }_i\right)}& \mathrm{Eq}.5\end{array}$
  where η is a reflection efficiency factor which is the value for x in the sin c²(x) function and where the value of sin c²(x) has decreased to a predetermined value from the maximum amplitude as indicated bypoints204,206 on thecurve200 inFIG. 24.
• The inventors have found that the reflection efficiency is acceptable when η≦1.39. This value for η corresponds to when the amplitude of the reflected beam (i.e., from the sin c²(x) function of Eqs. 3 & 4) has decayed to about 50% of its peak value. In particular, when x=1.39=η, sin c²(x)=0.5. However, other values for efficiency thresholds or factor in the Bragg envelope may be used if desired within the spirit of the invention.
• InFIG. 24, the beams26-36 are imaged onto theCCD camera60 to produce the pattern of light and dark regions122-132 inFIG. 25(a) representing a digital (or binary) code, where light=1 and dark=0 (or vice versa). The digital code may be generated by selectively creating individual index variations (or individual gratings) with the desired spatial periods Λ1-Λn. Other illumination, readout techniques, types of gratings, geometries, materials, etc. may be used as discussed in the aforementioned patent application.
• Referring toFIG. 26, illustrations (a)-(c), for the grating12 in acylindrical substrate10 having a sample spectral 17 bit code (i.e., 17 different pitches Λ1-Λ17), the corresponding image on the CCD (Charge Coupled Device)camera60 is shown for a digital pattern of 17bit locations89, includingFIG. 26, illustrations (a), (b) and (c), respectively, i.e. 7 bits turned on (10110010001001001); 9 bits turned on of (11000101010100111); and all 17 bits turned on of (11111111111111111).
• For the images inFIG. 26, the length of thesubstrate10 was 450 microns, the outer diameter D1 was 65 microns, the inner diameter D was 14 microns, δn for the grating12 was about 10⁻⁴, n1 inportion20 was about 1.458 (at a wavelength of about 1550 nm), n2 inportion18 was about 1.453, the average pitch spacing Λ for the grating12 was about 0.542 microns, and the spacing between pitches ΔΛ was about 0.36% of the adjacent pitches Λ.
• Referring toFIG. 27, illustration (a), the pitch Λ of an individual grating is the axial spatial period of the sinusoidal variation in the refractive index n1 in theregion20 of thesubstrate10 along the axial length of the grating12 as indicated by acurve90 on a graph91. Referring toFIG. 27, illustration (b), a sample composite grating12 comprises three individual gratings that are co-located on thesubstrate10, each individual grating having slightly different pitches, Λ1, Λ2, Λ3, respectively, and the difference (or spacing) ΔΛ between each pitch Λ being about 3.0% of the period of an adjacent pitch Λ as indicated by a series of curves92 on a graph94. Referring toFIG. 27, illustration (c), three individual gratings, each having slightly different pitches, Λ1, Λ2, Λ3, respectively, are shown, the difference ΔΛ between each pitch Λ being about 0.3% of the pitch Λ of the adjacent pitch as shown by a series ofcurves95 on agraph97. The individual gratings inFIG. 27, illustrations (b) and (c) are shown to all start at 0 for illustration purposes; however, it should be understood that the separate gratings need not all start in phase with each other. Referring toFIG. 27, illustration (d), the overlapping of the individual sinusoidal refractive index variation pitches Λ1-Λn in thegrating region20 of thesubstrate10 produces a combined resultant refractive index variation in thecomposite grating12 shown as acurve96 on a graph98 representing the combination of the three pitches shown inFIG. 27, illustration (b). Accordingly, the resultant refractive index variation in thegrating region20 of thesubstrate10 may not be sinusoidal and is a combination of the individual pitches Λ (or index variation).
• The maximum number of resolvable bits N, which is equal to the number of different grating pitches Λ (and hence the number of codes), that can be accurately read (or resolved) using side-illumination and side-reading of the grating12 in thesubstrate10, is determined by numerous factors, including: the beam width w incident on the substrate (and the corresponding substrate length L and grating length Lg), the thickness or diameter D of the grating12, the wavelength λ of incident light, the beam divergence angle θ_R, and the width of the Bragg envelope θ_B(discussed more in the aforementioned patent application), and may be determined by the equation:$\begin{array}{cc}N\cong \frac{\eta \beta L}{2D\sin \left({\theta }_i\right)}& \mathrm{Eq}.6\end{array}$
• Referring toFIG. 28, instead of having theinput light24 at a single wavelength λ (monochromatic) and reading the bits by the angle θo of the output light, the bits (or grating pitches Λ) may be read/detected by providing a plurality of wavelengths and reading the wavelength spectrum of the reflected output light signal. In this case, there would be one bit per wavelength, and thus, the code is contained in the wavelength information of the reflected output signal.
• In this case, each bit (or Λ) is defined by whether its corresponding wavelength falls within the Bragg envelope, not by its angular position within theBragg envelope200. As a result, it is not limited by the number of angles that can fit in theBragg envelope200 for a given composite grating12, as in the embodiment discussed hereinbefore. Thus, when using multiple wavelengths, the only limitation in the number of bits N is the maximum number of grating pitches Λ that can be superimposed and optically distinguished in wavelength space for the output beam.
• Referring toFIGS. 28 and 29, illustration (a), the reflection wavelength spectrum (λ1-λn) of the reflectedoutput beam310 will exhibit a series of reflection peaks695, each appearing at the same output Bragg angle θo. Each wavelength peak695 (λ1-λn) corresponds to an associated spatial period (Λ1-Λn), which make up thegrating12.
• One way to measure the bits in wavelength space is to have the input light angle θi equal to the output light angle θo, which is kept at a constant value, and to provide an input wavelength λ that satisfies the diffraction condition (Eq. 1) for each grating pitch Λ. This will maximize the optical power of the output signal for each pitch Λ detected in thegrating12.
• Referring to29, illustration (b), the transmission wavelength spectrum of the transmitted output beam330 (which is transmitted straight through the grating12) will exhibit a series of notches (or dark spots)696. Alternatively, instead of detecting the reflectedoutput light310, the transmitted light330 may be detected at the detector/reader308 (FIG. 28). It should be understood that the optical signal levels for the reflection peaks695 andtransmission notches696 will depend on the “strength” of the grating12, i.e., the magnitude of the index variation n in thegrating12.
• InFIG. 28, the bits may be detected by continuously scanning the input wavelength. A knownoptical source300 provides the inputlight signal24 of a coherent scanned wavelength input light shown as agraph304. Thesource300 provides a sync signal on aline306 to a knownreader308. The sync signal may be a timed pulse or a voltage ramped signal, which is indicative of the wavelength being provided as theinput light24 to thesubstrate10 at any given time. Thereader308 may be a photodiode, CCD camera, or other optical detection device that detects when an optical signal is present and provides an output signal on aline309 indicative of the code in thesubstrate10 or of the wavelengths present in the output light, which is directly related to the code, as discussed herein. The grating12 reflects theinput light24 and provides anoutput light signal310 to thereader308. The wavelength of the input signal is set such that the reflectedoutput light310 will be substantially in thecenter314 of theBragg envelope200 for the individual grating pitch (or bit) being read.
• Alternatively, thesource300 may provide a continuous broadband wavelength input signal such as that shown as agraph316. In that case, the reflectedoutput beam310 signal is provided to a narrowband scanning filter318 which scans across the desired range of wavelengths and provides a filtered outputoptical signal320 through alens321 to thereader308. Thescanning filter318 provides a sync signal on aline322 to the reader, which is indicative of which wavelengths are being provided on theoutput signal320 to the reader and may be similar to the sync signal discussed hereinbefore on theline306 from thesource300. In this case, thesource300 does not need to provide a sync signal because the inputoptical signal24 is continuous. Alternatively, instead of having the scanning filter being located in the path of theoutput beam310, the scanning filter may be located in the path of theinput beam24 as indicated by the dashedbox324, which provides the sync signal on aline323.
• Alternatively, instead of the scanning filters318,324, thereader308 may be a known optical spectrometer (such as a known spectrum analyzer), capable of measuring the wavelength of the output light.
• The desired values for the input wavelengths λ (or wavelength range) for theinput signal24 from thesource300 may be determined from the Bragg condition of Eq. 1, for a given grating spacing Λ and equal angles for the input light θi and the angle light θo. Solving Eq. 1 for λ and plugging in m=1, gives:
  λ=Λ[sin(θo)+sin(θi)]  Eq. 7
• It is also possible to combine the angular-based code detection with the wavelength-based code detection, both discussed hereinbefore. In this case, each readout wavelength is associated with a predetermined number of bits within the Bragg envelope. Bits (or grating pitches Λ) written for different wavelengths do not show up unless the correct wavelength is used.
• Accordingly, the scope of the invention is intended to include the bits (or grating pitches Λ) being read using one wavelength and many angles, many wavelengths and one angle, many wavelengths and many angles, or some combination thereof, as well as other techniques now known or later developed in the future. In other words, the scope of the invention is not intended to be limited to the number of wavelengths or angles used to read the bits of the code.
• Referring toFIG. 30, the grating12 may have a thickness or depth D which is comparable or smaller than the incident beam wavelength λ. This is known as a “thin” diffraction grating (or the full angle Bragg envelope is 180 degrees). In that case, the half-angle Bragg envelope θB is substantially 90 degrees; however, δn must be made large enough to provide sufficient reflection efficiency, per Eqs. 3 and 4. In particular, for a “thin” grating, D*δn_≈λ/2, which corresponds to a π phase shift between adjacent minimum and maximum refractive index values of the grating12.
• It should be understood that there is still a trade-off discussed hereinbefore with beam divergence angle θ_Rand the incident beam width (or length L of the substrate), but the accessible angular space is theoretically now 90 degrees. Also, for maximum efficiency, the phase shift between adjacent minimum and maximum refractive index values of the grating12 should approach a π phase shift; however, other phase shifts may be used.
• In this case, rather than having theinput light24 coming in at the conventional Bragg input angle θi, as discussed hereinbefore and indicated by a dashedline701, the grating12 is illuminated with the input light24 oriented on aline705 orthogonal to the longitudinalgrating vector705. Theinput beam24 will split into two (or more) beams of equal amplitude, where the exit angle θ_ocan be determined from Eq. 1 with the input angle θ_i=0 (normal to the longitudinal axis of the grating12).
• In particular, from Eq. 1, for a given grating pitch Λ1, the +/−1^storder beams (m=+1 and m=−1) corresponds tooutput beams700,702, respectively; the +/−2^ndorder beams (m=+2 and m=−2) corresponds tooutput beams704,706, respectively; and the 0^thorder (undiffracted) beam (m=0) corresponds tobeam708 and passes straight through the substrate. The output beams700-708 project spectral spots or peaks710-718, respectively, along a common plane, shown from the side by aline709, which is parallel to the upper surface of thesubstrate10.
• For example, for a grating pitch Λ=1.0 um, and an input wavelength λ=400 nm, the exit angles θ_oare ˜+/−23.6 degrees (for m=+/−1), and +/−53.1 degrees (from m=+/−2), from Eq. 1. It should be understood that for certain wavelengths, certain orders (e.g., m=+/−2) may be reflected back toward the input side or otherwise not detectable at the output side of the grating12.
• Alternatively, one can use only the +/−1^storder (m=+/−1) output beams for the code, in which case there would be only 2 peaks to detect,712,714. Alternatively, one can also use any one or more pairs from any order output beam that is capable of being detected. Alternatively, instead of using a pair of output peaks for a given order, an individual peak may be used.
• Referring toFIG. 31, if two pitches Λ1,Λ2 exist in the grating12, two sets of peaks will exist. In particular, for a second grating pitch Λ2, the +/−1^storder beams (m=+1 and m=−1) corresponds tooutput beams720,722, respectively; the +/−2^ndorder beams (m=+2 and m=−2) corresponds tooutput beams724,726, respectively; and the 0^thorder (un-diffracted) beam (m=0) corresponds tobeam718 and passes straight through the substrate. The output beams720-726 corresponding to the second pitch Λ2 project spectral spots or peaks730-736, respectively, which are at a different location than the point710-716, but along the same common plane, shown from the side by theline709.
• Thus, for a given pitch Λ (or bit) in a grating, a set of spectral peaks will appear at a specific location in space. Thus, each different pitch corresponds to a different elevation or output angle which corresponds to a predetermined set of spectral peaks. Accordingly, the presence or absence of a particular peak or set of spectral peaks defines the code.
• In general, if the angle of the grating12 is not properly aligned with respect to the mechanical longitudinal axis of thesubstrate10, the readout angles may no longer be symmetric, leading to possible difficulties in readout. With a thin grating, the angular sensitivity to the alignment of the longitudinal axis of thesubstrate10 to the input angle θi of incident radiation is reduced or eliminated. In particular, the input light can be oriented along substantially any angle θi with respect to the grating12 without causing output signal degradation, due the large Bragg angle envelope. Also, if theincident beam24 is normal to thesubstrate10, the grating12 can be oriented at any rotational (or azimuthal) angle without causing output signal degradation. However, in each of these cases, changing the incident angle θi will affect the output angle θo of the reflected light in a predetermined predictable way, thereby allowing for accurate output code signal detection or compensation.
• Referring toFIG. 32, for a thin grating, in addition to multiplexing in the elevation or output angle based on grating pitch Λ, the bits can also be multiplexed in an azimuthal (or rotational) angle θa of the substrate. In particular, a plurality ofgratings750,752,754,756 each having the same pitch Λ are disposed in asurface7011 of thesubstrate10 and located in the plane of thesubstrate surface7011. Theinput light24 is incident on all thegratings750,752,754,756 simultaneously. Each of the gratings provides output beams oriented based on the grating orientation. For example, thegrating750 provides the output beams762,764, thegrating752 provides the output beams766,768, thegrating754 provides the output beams770,772, and the grating756 provides the output beams774,776. Each of the output beams provides spectral peaks or spots (similar to that discussed hereinbefore), which are located in aplane760 that is parallel to thesubstrate surface plane7011. In this case, a single grating pitch Λ can produce many bits depending on the number of gratings that can be placed at different azimuthal (rotational) angles on the surface of thesubstrate10 and the number of output beam spectral peaks that can be spatially and optically resolved/detected. Each bit may be viewed as the presence or absence of a pair of peaks located at a predetermined location in space in theplane760. Note that this example uses only the m=+/−1^storder for each reflected output beam. Alternatively, the detection may also use the m=+/−2^ndorder. In that case, there would be two additional output beams and peaks (not shown) for each grating (as discussed hereinbefore) that may lie in the same plane as theplane760 and may be on a concentric circle outside thecircle760.
• In addition, the azimuthal multiplexing can be combined with the elevation or output angle multiplexing discussed hereinbefore to provide two levels of multiplexing. Accordingly, for a thin grating, the number of bits can be multiplexed based on the number of grating pitches Λ and/or geometrically by the orientation of the grating pitches.
• Furthermore, if the input light angle θi is normal to thesubstrate10, the edges of thesubstrate10 no longer scatter light from the incident angle into the “code angular space”, as discussed herein and/or in the aforementioned patent application.
• Also, in the thin grating geometry, a continuous broadband wavelength source may be used as the optical source if desired.
• Referring toFIG. 33, instead of or in addition to the pitches Λ in the grating12 being oriented normal to the longitudinal axis, the pitches may be created at an angle θg (also known as a blaze angle). In that case, theinput light24 incident normal to thesurface792 will produce a reflectedoutput beam790 having an angle θo determined by Eq. 1 as adjusted for the blaze angle θg. This can provide another level of multiplexing bits in the code.
• Referring toFIG. 34, instead of using an optical binary (0-1) code, an additional level of multiplexing may be provided by having an optical code using other numerical bases, if intensity levels of each bit are used to indicate code information. This could be achieved by having a corresponding magnitude (or strength) of the refractive index change (δn) for each grating pitch Λ. Four intensity ranges are shown for each bit number or pitch Λ, providing for a Base-4 code (where each bit corresponds to 0, 1, 2, or 3). The lowest intensity level, corresponding to a 0, would exist when this pitch Λ is not present in thegrating12. Thenext intensity level450 would occur when a first low level δn1 exists in the grating that provides an output signal within the intensity range corresponding to a 1. The next intensity level452 would occur when a second higher level δn2 exists in the grating12 that provides an output signal within the intensity range corresponding to a 2. The next intensity level454, would occur when a third higher level δn3 exists in the grating12 that provides an output signal within the intensity range corresponding to a 3.
• Referring toFIG. 35, theinput light24 may be incident on thesubstrate10 on an end face600 of thesubstrate10. In that case, theinput light24 will be incident on the grating12 having a more significant component of the light (as compared to side illumination discussed hereinbefore) along the longitudinalgrating axis207 of the grating (along the grating vector k_B), as shown by a line602. The light602 reflects off the grating12 as indicated by a line604 and exits the substrate as output light608. Accordingly, it should be understood by one skilled in the art that the diffraction equations discussed hereinbefore regarding output diffraction angle θo also apply in this case except that the reference axis would now be thegrating axis207. Thus, in this case, the input and output light angles θi,θo, would be measured from thegrating axis207 and length Lg of the grating12 would become the thickness or depth D of the grating12. As a result, a grating12 that is 400 microns long, would result in theBragg envelope200 being narrow. It should be understood that because the values of n1 and n2 are close to the same value, the slight angle changes of the light between theregions18,20 are not shown herein.
• In the case where incident light610 is incident along the same direction as the grating vector (Kb)207, i.e., θi=0 degrees, the incident light sees the whole length Lg of the grating12 and the grating provides a reflected output light angle θo=0 degrees, and the Bragg envelope612 becomes extremely narrow, as the narrowing effect discussed above reaches a limit. In that case, the relationship between a given pitch Λ in the grating12 and the wavelength of reflection λ is governed by a known “Bragg grating” relation:
  λ=2n_effΛ  Eq. 8
  where n_effis the effective index of refraction of the substrate, λ is the input (and output wavelength) and Λ is the pitch. This relation, as is known, may be derived from Eq. 1 where θi=θo=90 degrees.
• In that case, the code information is readable only in the spectral wavelength of the reflected beam, similar to that discussed hereinbefore for wavelength based code reading. Accordingly, the input signal in this case may be a scanned wavelength source or a broadband wavelength source. In addition, as discussed hereinbefore for wavelength based code reading, the code information may be obtained in reflection from the reflected beam614 or in transmission by the transmitted beam616 that passes through thegrating12.
• It should be understood that for shapes of thesubstrate10 orelement8 other than a cylinder, the effect of various different shapes on the propagation of input light through theelement8,substrate10, and/or grating12, and the associated reflection angles, can be determined using known optical physics including Snell's Law, shown below:
  n_insin θ_{in =}n_outsin θ_out  Eq. 9
• where n_inis the refractive index of the first (input) medium, and n_outis the refractive index of the second (output) medium, and θin and θout are measured from a line620 normal to the incident surface600.
• Referring toFIG. 36, if the value of n1 in thegrating region20 is greater than the value of n2 in thenon-grating region18, thegrating region20 of thesubstrate10 will act as a known optical waveguide for certain wavelengths. In that case, thegrating region20 acts as a “core” along which light is guided and theouter region18 acts as a “cladding” which helps confine or guide the light. Also, such a waveguide will have a known “numerical aperture” (θna) that will allow light630 that is within the aperture θna to be directed or guided along thegrating axis207 and reflected axially off the grating12 and returned and guided along the waveguide. In that case, the grating12 will reflect light632 having the appropriate wavelengths equal to the pitches Λ present in the grating12 back along the region20 (or core) of the waveguide, and pass the remaining wavelengths of light as the light632′. Thus, having thegrating region20 act as an optical waveguide for wavelengths reflected by the grating12 allows incident light that is not aligned exactly with thegrating axis207 to be guided along and aligned with the grating12axis207 for optimal grating reflection.
• If an optical waveguide is used any standard waveguide may be used, e.g., a standard telecommunication single mode optical fiber (125 micron diameter or 80 micron diameter fiber with about a 8-10 micron diameter), or a larger diameter waveguide (greater than 0.5 mm diameter), such as is describe in U.S. patent application Ser. No. 09/455,868, filed Dec. 6, 1999, entitled “Large Diameter Waveguide, Grating”. Further, any type of optical waveguide may be used for theoptical substrate10, such as, a multi-mode, birefringent, polarization maintaining, polarizing, multi-core, multi-cladding, or microsturctured optical waveguide, or a flat or planar waveguide (where the waveguide is rectangular shaped), or other waveguides.
• Referring toFIG. 37, if the grating12 extends across the entire dimension D1 (FIG. 2) of thesubstrate10, thesubstrate10 does not behave as a waveguide for the incident or reflected light and theincident light24 will be diffracted (or reflected) as indicated bylines642, and the codes detected as discussed hereinbefore for the end-incidence condition discussed hereinbefore withFIG. 45, and the remaining light640 passes straight through.
• Referring toFIG. 38, illustrations (a)-(c), in illustration (a), for the end illumination condition, if a blazed or angled grating is used, as discussed hereinbefore, theinput light24 is coupled out of thesubstrate10 at a known angle as shown by aline650. Referring toFIG. 38, illustration (b), alternatively, theinput light24 may be incident from the side and, if the grating12 has the appropriate blaze angle, the reflected light will exit from theend face652 as indicated by aline654. Referring toFIG. 38, illustration (c), the grating12 may have a plurality of different pitch angles660,662, which reflect theinput light24 to different output angles as indicated bylines664,666. This provides another level of multiplexing (spatially) additional codes, if desired.
• The grating12 may be impressed in thesubstrate10 by any technique for writing, impressed, embedded, imprinted, or otherwise forming a diffraction grating in the volume of or on a surface of asubstrate10. Examples of some known techniques are described in U.S. Pat. Nos. 4,725,110 and 4,807,950, entitled “Method for Impressing Gratings Within Fiber Optics”, to Glenn et al; and U.S. Pat. No. 5,388,173, entitled “Method and Apparatus for Forming Aperiodic Gratings in Optical Fibers”, to Glenn, respectively, and U.S. Pat. No. 5,367,588, entitled “Method of Fabricating Bragg Gratings Using a Silica Glass Phase Grating Mask and Mask Used by Same”, to Hill, and U.S. Pat. No. 3,916,182, entitled “Periodic Dielectric Waveguide Filter”, Dabby et al, and U.S. Pat. No. 3,891,302, entitled “Method of Filtering Modes in Optical Waveguides”, to Dabby et al, which are all incorporated herein by reference to the extent necessary to understand the present invention.
• Alternatively, instead of the grating12 being impressed within the substrate material, the grating12 may be partially or totally created by etching or otherwise altering the outer surface geometry of the substrate to create a corrugated or varying surface geometry of the substrate, such as is described in U.S. Pat. No. 3,891,302, entitled “Method of Filtering Modes in Optical Waveguides”, to Dabby et al, which is incorporated herein by reference to the extent necessary to understand the present invention, provided the resultant optical refractive profile for the desired code is created.
• Further, alternatively, the grating12 may be made by depositing dielectric layers onto the substrate, similar to the way a known thin film filter is created, so as to create the desired resultant optical refractive profile for the desired code.
FIGS.39-50: Alternative Microbead Geometries
• FIGS. 39-50 set forth microbead geometries consistent with that shown inFIGS. 2-3.
• For example, the scope of the invention is intended to include the substrate10 (and/or the element8) having end-view cross-sectional shapes other than circular, such as square, rectangular, elliptical, clam-shell, D-shaped, or other shapes, and may have side-view sectional shapes other than rectangular, such as circular, square, elliptical, clam-shell, D-shaped, or other shapes. Also, 3D geometries other than a cylinder may be used, such as a sphere, a cube, a pyramid or any other 3D shape. Alternatively, thesubstrate10 may have a geometry that is a combination of one or more of the foregoing shapes.
• The shape of theelement8 and the size of the incident beam may be made to minimize any end scatter off the end face(s) of theelement8, as is discussed herein and/or in the aforementioned patent application. Accordingly, to minimize such scatter, theincident beam24 may be oval shaped where the narrow portion of the oval is smaller than the diameter D1, and the long portion of the oval is smaller than the length L of theelement8. Alternatively, the shape of the end faces may be rounded or other shapes or may be coated with an antireflective coating.
• It should be understood that the size of any given dimension for theregion20 of the grating12 may be less than any corresponding dimension of thesubstrate10. For example, if the grating12 has dimensions of length Lg, depth Dg, and width Wg, and thesubstrate12 has different dimensions of length L, depth D, and width W, the dimensions of the grating12 may be less than that of thesubstrate12. Thus, the grating12 may be embedded within or part of a muchlarger substrate12. Also, theelement8 may be embedded or formed in or on a larger object for identification of the object.
• The dimensions, geometries, materials, and material properties of thesubstrate10 are selected such that the desired optical and material properties are met for a given application. The resolution and range for the optical codes are scalable by controlling these parameters as discussed herein and/or in the aforementioned patent application.
• Referring toFIG. 39, thesubstrate10 may have anouter coating799, such as a polymer or other material that may be dissimilar to the material of thesubstrate10, provided that thecoating799 on at least a portion of the substrate allows sufficient light to pass through the substrate for adequate optical detection of the code. Thecoating799 may be on any one or more sides of thesubstrate10. Also, thecoating799 may be a material that causes theelement8 to float or sink in certain fluids (liquid and/or gas) solutions.
• Also, thesubstrate10 may be made of a material that is less dense than certain fluid (liquids and/or gas) solutions, thereby allowing theelements8 to float or be buoyant or partially buoyant. Also, the substrate may be made of a porous material, such as controlled pore glass (CPG) or other porous material, which may also reduce the density of theelement8 and may make theelement8 buoyant or partially-buoyant in certain fluids.
• Referring toFIG. 40, the grating12 is axially spatially invariant. As a result, thesubstrate10 with the grating12 (shown as a long substrate21) may be axially subdivided or cut into many separatesmaller substrates30′,32′,34′,36′ and eachsubstrate30′-36′ will contain the same code as thelonger substrate21 had before it was cut. The limit on the size of thesmaller substrates30′-36′ is based on design and performance factors discussed herein and/or in the aforementioned patent application.
• Referring toFIG. 41, one purpose of the outer region18 (or region without the grating12) of thesubstrate10 is to provide mechanical or structural support for the innergrating region20. Accordingly, theentire substrate10 may comprise the grating12, if desired. Alternatively, the support portion may be completely or partially beneath, above, or along one or more sides of thegrating region20, such as in a planar geometry, or a D-shaped geometry, or other geometries, as described herein and/or in the aforementioned patent application. Thenon-grating portion18 of thesubstrate10 may be used for other purposes as well, such as optical lensing effects or other effects (discussed herein or in the aforementioned patent application). Also, the end faces of thesubstrate10 need not be perpendicular to the sides or parallel to each other. However, for applications where theelements8 are stacked end-to-end, the packing density may be optimized if the end faces are perpendicular to the sides.
• Referring toFIG. 42, illustrations (a)-(c), two ormore substrates10,250, each having at least one grating therein, may be attached together to form theelement8, e.g., by an adhesive, fusing or other attachment techniques. In that case, thegratings12,252 may have the same or different codes.
• Referring toFIG. 43, illustrations (a) and (b), thesubstrate10 may havemultiple regions80,90 and one or more of these regions may have gratings in them. For example, there may begratings12,252 side-by-side (illustration (a)), or there may be gratings82-88, spaced end-to-end (illustration (b)) in thesubstrate10.
• Referring toFIG. 44, the length L of theelement8 may be shorter than its diameter D, thus, having a geometry such as a plug, puck, wafer, disc or plate.
• Referring toFIG. 45 to facilitate proper alignment of the grating axis with the angle θi of theinput beam24, thesubstrate10 may have a plurality of thegratings12 having the same codes written therein at numerous different angular or rotational (or azimuthal) positions of thesubstrate10. In particular, twogratings550,552, having axialgrating axes551,553, respectively may have a common central (or pivot or rotational) point where the twoaxes551,553 intersect. The angle θi of theincident light24 is aligned properly with the grating550 and is not aligned with the grating552, such that output light555 is reflected off thegrating550 and light557 passes through the grating550 as discussed herein. If theelement8 is rotated as shown by the arrows559, the angle θi of incident light24 will become aligned properly with the grating552 and not aligned with the grating550 such that output light555 is reflected off thegrating552 and light557 passes through thegrating552. When multiple gratings are located in this rotational orientation, the bead may be rotated as indicated by a line559 and there may be many angular positions that will provide correct (or optimal) incident input angles θi to the grating. While this example shows a circular cross-section, this technique may be used with any shape cross-section.
• Referring toFIG. 46, illustrations (a), (b), (c), (d), and (e) thesubstrate10 may have one or more holes located within thesubstrate10. In illustration (a), holes560 may be located at various points along all or a portion of the length of thesubstrate10. The holes need not pass all the way through thesubstrate10. Any number, size and spacing for the holes560 may be used if desired. In illustration (b), holes572 may be located very close together to form a honeycomb-like area of all or a portion of the cross-section. In illustration (c), one (or more)inner hole566 may be located in the center of thesubstrate10 or anywhere inside of where the grating region(s)20 are located. Theinner hole566 may be coated with areflective coating573 to reflect light to facilitate reading of one or more of thegratings12 and/or to reflect light diffracted off one or more of thegratings12. Theincident light24 may reflect off the grating12 in theregion20 and then reflect off thesurface573 to provideoutput light577. Alternatively, theincident light24 may reflect off thesurface573, then reflect off the grating12 and provide theoutput light575. In that case thegrating region20 may run axially or circumferentially571 around thesubstrate10. In illustration (d), the holes579 may be located circumferentially around thegrating region20 or transversely across thesubstrate10. In illustration (e), the grating12 may be located circumferentially around the outside of thesubstrate10, and there may beholes574 inside thesubstrate10.
• Referring toFIG. 47, illustrations (a), (b), and (c), thesubstrate10 may have one or more protruding portions orteeth570,578,580 extending radially and/or circumferentially from thesubstrate10. Alternatively, theteeth570,578,580 may have any other desired shape.
• Referring toFIG. 48, illustrations (a), (b), (c) a D-shaped substrate, a flat-sided substrate and an eye-shaped (or clam-shell or teardrop shaped)substrate10, respectively, are shown. Also, thegrating region20 may have end cross-sectional shapes other than circular and may have side cross-sectional shapes other than rectangular, such as any of the geometries described herein for thesubstrate10. For example, thegrating region20 may have a oval cross-sectional shape as shown by dashedlines581, which may be oriented in a desired direction, consistent with the teachings herein. Any other geometries for thesubstrate10 or thegrating region20 may be used if desired, as described herein.
• Referring toFIG. 49, at least a portion of a side of thesubstrate10 may be coated with areflective coating514 to allow incident light510 to be reflected back to the same side from which the incident light came, as indicated by reflectedlight512.
• Referring toFIG. 50, illustrations (a) and (b), alternatively, thesubstrate10 can be electrically and/or magnetically polarized, by a dopant or coating, which may be used to ease handling and/or alignment or orientation of thesubstrate10 and/or the grating12, or used for other purposes. Alternatively, the bead may be coated with conductive material, e.g., metal coating on the inside of a holey substrate, or metallic dopant inside the substrate. In these cases, such materials can cause thesubstrate10 to align in an electric or magnetic field. Alternatively, the substrate can be doped with an element or compound that fluoresces or glows under appropriate illumination, e.g., a rare earth dopant, such as Erbium, or other rare earth dopant or fluorescent or luminescent molecule. In that case, such fluorescence or luminescence may aid in locating and/or aligning substrates.
• InFIGS. 51 and 52,beads8 are shown being read from flat retro-reflector trays200(a) and200(b) respectively in accordance with the present invention. InFIG. 52, the tray200(b) has a reflective coating2050 for reflecting light27 reflecting off the grating12 as reflected light27′ as shown.
• InFIGS. 53 and 54, beads are shown being read through V-grooves205 of thetray200, consistent with that shown inFIG. 4.
FIGS.55-65: CV-0053
• FIGS.55 to65 show alternative embodiments which are related and form part of the subject matter of the present application U.S. patent application Ser. No. 10/661,836 filed Sep. 12, 2003, which is hereby incorporated by reference in its entirety.
FIG.55
• In particular, FIGS.55(a), (b),(c) and (d) show an embodiment of the present invention that uses microtitre plates whose bottoms consist of flat or contoured groove plates. In this embodiment, beads are agitated to align themselves in the groove plates, and then codes and labels are read directly from the bottom of the microtitre plates with standard or custom fluorescence scanners. This embodiment has the advantage of removing the bead transfer step, which in turn increases sample throughput and reduces the requisite investment in fluidic handling automation equipment. InFIG. 55(b), themicrotitre plate1900 hasstraight grooves1902 in the bottom that are about as wide as they are deep (1:1 aspect ratio) and can have a pitch of 1 to 200 microns. In comparison, inFIG. 55(d), themicrotitre plate1910 hascircular grooves1912 arranged concentrically. In addition, the grooves may be 100% to 200% of the bead diameter, and preferably about 120%. For example, if the bead diameter is 28 microns, then the groove diameter should be about 32 microns. Moreover, it is important to note that the scope of the invention is not intended to be limited only to straight or concentric circular grooves, since embodiments are envisioned using grooves having other geometries, including diamond shaped, swervy, etc.
FIGS.56-60
• FIGS. 56-60 show a cell or tray design for loading and unloading beads during the bead alignment and interrogation process. The design makes bead loading/unloading more convenient. The goals are to be able to pipette the beads into the cell or tray; easily vacuum or wash the beads from the cell or tray; easily disassemble and clean the cell or tray; and provide an open cell architecture that lets bubbles out but keeps beads wet for hours.
FIGS.56(a) and (b)
• FIGS.56(a) and (b) show a side and top view of a cell ortray2100 having four sides surrounding a glass plate or bottom2102 withgrooves2104, and having a top2106 hingeably coupled to the cell ortray2100.
FIGS.57(a) and (b): Bead Loading
• FIGS.57(a) and (b) show bead loading, withFIG. 57(a) showing the top open2106 and a pipette2110 for providingbeads8 into the tray orcell2100 so as to fill it like a so-called “pond”. The cell ortray2100 is stimulated, for example, using a magnetic stir bar, to aid the alignment of thebeads8 in thegrooves2104 on thebottom plate2102. InFIG. 57(b), the top2106 is closed when thebeads8 are aligned.
FIGS.58(a) and (b): Bead Unloading
• FIGS.58(a) and (b) show bead unloading, withFIG. 58(a) showing the top2106 opening, andFIG. 58(b) showing avacuum2112 sucking thebeads8 out. Alternatively, the cell ortray2100 can be turned sideways and thebeads8 flushed out with a jet of liquid from awater providing device2114.
FIGS.59-60
• FIG. 59 shows an elevated perspective of the cell ortray2100 with the cover down, whileFIG. 60 shows an elevated perspective of the cell ortray2100 with thecover2106 up.
FIG.61: Bead Alignment Device Having Narrow Groove Plate
• FIG. 61 shows an alternative embodiment in which thebead alignment device2200 has anarrow window2216. As shown, the grooves2204 are in a section2218 of the bottom plate, not across entire bottom. In response to some stimulation, the beads (not shown) are corralled innarrow window2016 and fall into alignment.
FIGS.62-64: Bead Alignment Device Having Wide Window
• FIG. 62 shows still another alternative embodiment generally indicated2300, in which the grooves2304 extend substantially along the entire base. When the solution having the microbeads (not shown) is provided to thebead alignment device2300, the beads align themselves in the grooves. Consistent with that discussed above, typically the bead alignment device is stimulated to encourage bead movement into and alignment in the grooves.
FIGS.65(a), (b), (c)
• FIG. 65(a), (b) and (c) show an alternative embodiment of the alignment cell or tray in the form of a test tube orother container2400,2400′. InFIG. 65(a), thegrooves2404 are arranged in the bottom of the test tube for aligning the beads contained in the liquid. InFIG. 65(b), the container is shown having multiple levels ofgrooved platforms2420,2422,2424,2426, which may be perforated with holes to allow liquid and/or thebeads8 to flow between levels.FIG. 65(c) shows top views of various containers, which may have different geometric shapes, including circular, oval, square, rectangular. The scope of the invention is not intended to be limited to any particular container shape.
The Scope of the Invention
• Unless otherwise specifically stated herein, the term “microbead” is used herein as a label and does not restrict any embodiment or application of the present invention to certain dimensions, materials and/or geometries.
• The dimensions and/or geometries for any of the embodiments described herein are merely for illustrative purposes and, as such, any other dimensions and/or geometries may be used if desired, depending on the application, size, performance, manufacturing requirements, or other factors, in view of the teachings herein.
• It should be understood that, unless stated otherwise herein, any of the features, characteristics, alternatives or modifications described regarding a particular embodiment herein may also be applied, used, or incorporated with any other embodiment described herein. Also, the drawings herein are not drawn to scale.
• Although the invention has been described and illustrated with respect to exemplary embodiments thereof, the foregoing and various other additions and omissions may be made therein and thereto without departing from the spirit and scope of the present invention.

Claims (69)

1. A method for aligning microbeads to be read by a code reading or other detection device, comprising the step of:

providing microbeads to a positioning device, each having an elongated body with a code embedded therein along a longitudinal axis thereof; and

aligning the microbeads with the positioning device so the longitudinal axis of the microbeads is in a fixed orientation relative to a code reading or other detection device.

2. A method according toclaim 1, wherein the positioning device is a plate having a multiplicity of grooves therein.

3. A method according toclaim 2, wherein the method includes agitating the plate to encourage the alignment of the microbeads in the grooves.

4. A method according toclaim 1, wherein the microbeads are cylindrically shaped glass beads between 25 and 250 microns in diameter and between 100 and 500 microns long.

5. A method according toclaim 1, wherein the microbeads have a holographic code embedded in a central region thereof.

6. A method according toclaim 1, wherein the code is used to correlate a chemical content on each bead with a measured fluorescence signal.

7. A method according toclaim 1, wherein each microbead is substantially aligned in relation to its pitch and yaw rotational axes.

8. A method according toclaim 1, wherein the positioning device has a series of parallel grooves having one of several different shapes, including square, v-shaped or semi-circular.

9. A method according toclaim 1, wherein the positioning device is an optically transparent medium including borosilicate glass, fused silica or plastic, and the grooves are formed therein.

10. A method according toclaim 2, wherein the grooves have a depth that is dimensioned to be at least the diameter of the microbeads, including at least 110% of the diameter of the microbead.

11. A method according toclaim 2, wherein either the grooves have a depth between 10 and 125 microns, the depth is dimensioned within 90% of the diameter of the microbeads, or a combination thereof.

12. A method according toclaim 2, wherein the spacing of the grooves is between 1 and 2 times the diameter of the microbeads.

13. A method according toclaim 2, wherein the grooves have a width that is dimensioned to prevent the beads from rotating therein by more than a few degrees.

14. A method according toclaim 2, wherein the grooves have a width that is dimensioned within 5% of the diameter of the microbeads.

15. A method according toclaim 2, wherein the grooves have a bottom that is flat enough to prevent the beads from rotating, by more than a few tenths of a degree, relative to the code reader device.

16. A method according toclaim 1, wherein the code reader device includes a readout camera.

17. A method according toclaim 3, wherein the step of agitating the plate includes using a sonic transducer, a mechanical wipe, or shaking or rocking device.

18. A method according toclaim 2, wherein the method includes using an open format approach by dispensing the microbeads onto the plate using a pipette tip or syringe tip and not covering the plate.

19. A method according toclaim 2, wherein the method includes a closed format approach by dispensing the microbeads into a cuvette-like device comprising the plate, at least three walls and a cover.

20. A method according toclaim 19, wherein the step of dispensing includes injecting the microbeads into the cuvette-like device by placing them near an edge of an opening and allowing the surface tension, or an induced fluid flow, to pull the microbeads into the cuvette-like device.

21. A method according toclaim 19, wherein the method includes using a closed format approach by sectioning a closed region into two regions, a first region where the microbeads are free to move about in a plane, either in a groove or not, and a second region where the microbeads are trapped in a groove and can only move along the axes of the grooves.

22. A method according toclaim 21, wherein the method includes the step of trapping the microbeads in a groove by reducing the height of the closed region so that the microbeads can no longer come out of the groove.

23. A method according toclaim 21, wherein the first region is used to pre-align the beads into a groove, facilitating the introduction of beads into the second region.

24. A method according toclaim 21, wherein the method includes tilting the cuvette-like up so gravity can be used to pull the microbeads along a groove from the first region to the second region.

25. A method according toclaim 21, wherein the plate is made of silicon having walls formed by Su8 coupled thereto, or having walls formed by etching the silicon.

26. A method according toclaim 1, wherein the method includes the step of identifying a chemical content on the surface of the microbead with a measured fluorescence signal.

27. A method according toclaim 1, wherein the method includes passing a code reading signal through the microbead aligned on the positioning device.

28. A method according toclaim 1, wherein the method further includes the step of correlating a chemical content identified on each microbead with a fluorescence signal, including one provided by an incident laser beam device.

29. A method according toclaim 1, wherein the method includes the step of identifying the code in the microbead.

30. A method according toclaim 2, wherein the grooves of the plate are formed using a photolithographic process.

31. A method according toclaim 2, wherein the plate includes a glass plate having Su8 thereon.

32. A method according toclaim 2, wherein the plate is made of a low fluorescence glass.

33. A method according toclaim 2, wherein the plate is made of a borosilicate glass.

34. A method according toclaim 2, wherein the grooves on the plate are mechanically machined.

35. A method according toclaim 2, wherein the grooves on the plate are formed by deep reactive ion etching.

36. A method according toclaim 2, wherein the grooves on the plate are formed by injection molding.

37. A method according toclaim 2, wherein the plate has a mirror coating.

38. A method according toclaim 2, wherein the plate is a disk having circumferential grooves, concentric grooves, or a combination thereof.

39. A method according toclaim 2, wherein the plate is a disk having radial grooves.

40. A method according toclaim 2, wherein the plate is a disk having a microbead loading area located in the center of the disk.

41. A method according toclaim 2, wherein the plate is a disk having one or more radial water channels extending from the center to the outer periphery thereof.

42. A method according toclaim 2, wherein the method includes arranging the plate on a rotating disk.

43. A method according toclaim 1, wherein the positioning device is a flow tube.

44. A method according toclaim 43, wherein the step of providing includes providing the microbeads to a flow tube in a fluid.

45. A method according toclaim 1, wherein the microbeads have tubular holes extending therethrough.

46. A method according toclaim 1, wherein the microbeads have teeth or protrusions thereon.

47. Apparatus for aligning microbeads to be read by a code reading or other detection device, comprising:

a positioning device for aligning microbeads, each microbead having an elongated body with a code embedded therein along a longitudinal axis thereof, so the longitudinal axis of the microbeads is positioned in a fixed orientation relative to the code reading device.

48. Apparatus according toclaim 47, wherein the positioning device is a plate having a multiplicity of grooves therein.

49. Apparatus according toclaim 48, wherein the apparatus includes means for agitating the plate to encourage the alignment of the microbeads in the grooves.

50. Apparatus according toclaim 47, wherein the microbeads are cylindrically shaped glass beads between 25 and 250 microns in diameter and between 100 and 500 microns long.

51. Apparatus according toclaim 47, wherein the microbeads have a holographic code embedded in a central region thereof.

52. Apparatus according toclaim 47, wherein the positioning device is a rotating disk having a multiplicity of circumferential grooves, concentric grooves or a combination thereof formed therein, or having one or more spiral grooves.

53. Apparatus according toclaim 47, wherein the positioning device is a tube.

54. Apparatus for aligning an optical identification element, comprising:

the optical identification element having an optical substrate having at least a portion thereof with at least one diffraction grating disposed therein, the grating having at least one refractive index pitch superimposed at a common location, said grating being embedded within a substantially single material of said substrate the grating providing an output optical signal when illuminated by an incident light signal propagating in free space, the optical output signal being indicative of a code, the output signal being the result of passive, non-resonant scattering from said at least one diffraction grating when illuminated by said incident light signal, and the optical identification element being an elongated object with a longitudinal axis; and

an alignment device which aligns the optical identification element such that said output optical signal is indicative of the code.

55. Apparatus according toclaim 54, wherein the alignment device is a plate having a multiplicity of grooves therein.

56. Apparatus according toclaim 55, wherein the plate is a disk and the multiplicity of grooves are concentric circles or a spiral.

57. Apparatus according toclaim 54, wherein the alignment device is a tube having a bore for receiving the optical identification element.

58. A method according toclaim 2, wherein each groove has a pitch in a range of 1-200 microns.

59. A method according toclaim 2, wherein each groove has a width and depth that are about the same.

60. A method according toclaim 2, wherein each groove is about 100%-200% of the diameter of the microbeads.

61. A method according toclaim 2, wherein each groove is about 120% of the diameter of the microbeads.

62. A method according toclaim 2, wherein the plate has a series of concentric grooves.

63. A method according toclaim 1, wherein the positioning device includes a base having a plate with grooves therein.

64. A method according toclaim 1, wherein the positioning device includes a cover for covering the same.

65. A method according toclaim 64, wherein the cover is coupled to the base by a hinge.

66. A method according toclaim 1, wherein the positioning device includes a container having a base with grooves therein.

67. A method according toclaim 1, wherein the positioning device includes a container having a multiple platforms, each with grooves therein.

68. A method according toclaim 67, wherein one or more of the multiple platforms has perforations for passing liquid, microbeads or a combination thereof.

69. A method according toclaim 67, wherein the container is circular, square, rectangular, or other suitable shape.

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